Anti-hypertensive agents

ABSTRACT

Novel compounds are disclosed as potent inhibitors of angiotensin converting enzyme and as orally effective antihypertensive agents. The compounds have the general formula: ##STR1##

BACKGROUND OF THE INVENTION

Angiotensin converting enzyme (peptidyldipeptide hydrolase, hereinafterreferred to as ACE) occupies a central role in the physiology ofhypertension. The enzyme is capable of converting the decapeptideangiotensin I, having the sequence

AspArgValTyrIleHisProPheHisLeu to an octapeptide, angiotensin II byremoval of the carboxyterminal HisLeu. The symbols for various chemicalentities are explained in the following table:

Ala=L-alanine

Arg=L-arginine

Asp=L-aspartic acid

<Glu=pyro-L-glutamic acid

Gly=glycine

Hip=Hippuric acid (Benzoyl glycine)

His=L-histidine

Ile=L-isoleucine

Leu=L-leucine

Phe=L-phenylalanine

Pro=L-proline

ΔPro=L-3,4-dehydroproline

Ser=L-serine

Trp=L-tryptophan

Tyr=L-tyrosine

Val=L-valine

ACE=Angiotensin converting enzyme

Hepes=N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

Angiotensin I is formed by the action of the enzyme renin, anendopeptidase found in kidney, other tissues and plasma, acting on aserum α-2 globulin.

Blood pressure is affected by certain peptides found in the blood. Oneof these, angiotensin II, is a powerful pressor (blood pressureelevating) agent. Another, bradykinin, a nonapeptide with the sequenceArgProProGlyPheSerProPheArg is a powerful depressor (blood pressurelowering) agent. In addition to a direct pressor effect, angiotensin IIstimulates release of aldosterone which tends to elevate blood pressureby causing retention of extracellular salt and fluids. Angiotensin II isfound in measurable amount in the blood of normal humans. However, it isfound at elevated concentrations in the blood of patients with renalhypertension.

The level of ACE activity is ordinarily in excess, in both normal andhypertensive humans, of the amount needed to maintain observed levels ofangiotensin II. However, it has been found that significant bloodpressure lowering is achieved in hypertensive patients by treatment withACE inhibitors. [Gavras, I., et al., New Engl. J. Med. 291, 817 (1974)].

ACE is a peptidyldipeptide hydrolase. It catalyzes the hydrolysis of thepenultimate peptide bond at the C-terminal end of a variety of acylatedtripeptides and larger polypeptides having an unblocked α-carboxylgroup. The action of ACE results in hydrolytic cleavage of thepenultimate peptide bond from the carboxyl-terminal end yielding asreaction products a dipeptide and a remnant.

The reactivity of the enzyme varies markedly depending on the substrate.At least one type of peptide bond, having the nitrogen supplied byproline, is not hydrolyzed at all. The apparent Michaelis constant (Km)varies from substrate to substrate over several orders of magnitude. Forgeneral discussion of the kinetic parameters of enzyme catalyzedreactions, see Lehninger, A., Biochemistry, Worth Publishers, Inc., NewYork, 1970, pp. 153-157. Many peptides which are called inhibitors ofthe enzymatic conversion of angiotensin I to angiotensin II are in factsubstrates having a lower Km than angiotensin I. Such peptides are moreproperly termed competitive substrates. Examples of competitivesubstrates include bradykinin, and the peptide BPP_(5a) (also calledSQ20475) from snake venom, whose sequence is <GluLysTrpAlaPro.

Numerous synthetic peptide derivatives have been shown to be ACEinhibitors by Ondetti, et al. in U.S. Pat. No. 3,832,337 issued Aug. 27,1974.

The role of ACE in the pathogenesis of hypertension has prompted asearch for inhibitors of the enzyme that could act as antihypertensivedrugs. See for example U.S. Pat. Nos. 3,891,616, 3,947,575, 4,052,511and 4,053,651. A highly effective inhibitor, with high biologicalactivity when orally administered, isD-3-mercapto-2-methylpropanoyl-L-proline, designated SQ14225, disclosedin U.S. Pat. No. 4,046,889 to Ondetti et al., issued Sept. 6, 1977, andin scientific articles by Cushman, D.W. et al., Biochemistry 16, 5484(1977), and by Ondetti, M. et al., Science 196, 441 (1977). Theinhibitor SQ14225 reportedly has an I₅₀ value of 2.3×10⁻⁸ M. The I₅₀value reported by Cushman, et al, supra is the concentration ofinhibitor required to produce 50% inhibition of the enzyme under astandard assay system containing substrate at a level substantiallyabove K_(m). It will be understood that I₅₀ values are directlycomparable when all potential factors affecting the reaction are keptconstant. These factors include the source of enzyme, its purity, thesubstrate used and its concentration, and the composition of the assaybuffer. All I₅₀ data reported herein have been performed with the sameassay system and same enzyme (human urinary ACE) and with anapproximately 1/2K_(m) level of substrate and are therefore internallyconsistent. Discrepancies with data obtained by other workers may beobserved. Indeed such discrepancies do exist in the literature, forunknown reasons. See, for example, the I₅₀ for BPP_(9a) reported byCushman, D. W., et al., Experientia 29, 1032 (1973) and by Dorer, F. E.,et al., Biochim.Biophys.Acta 429, 220 (1976).

The mode of action of SQ 14,225 has been based upon a model of theactive site of ACE developed by analogy with the better known relatedenzyme, carboxypeptidase A. The active site was postulated to have acationic site for binding the carboxyl end group of the substrate and apocket or cleft capable of binding the side chain of the C-terminalamino acid and providing especially tight binding for the heterocyclicring of a terminal proline residue. A similar pocket for the penultimateamino acid residue was postulated, and the published data suggested arather stringent steric requirement, since the D-form of the inhibitorwas substantially more potent than its stereoisomer or the 3-methyl andunsubstituted analogs. The sulfhydryl group on the inhibitor, postulatedto be bound at the active site near the catalytic center, was believedto play a central role in inactivation of the enzyme by combining withthe zinc moiety known to be essential for catalytic activity.Substituents on the sulfhydryl, such as a methyl group, and an S-acetylderivative, substantially reduced potency of the inhibitor. See Cushman,D. W., et al., Biochemistry, supra.

In vitro study of the mechanism by which SQ 14,225 and its analogs actto inhibit ACE has been somewhat hampered by the instability of thesemolecules under ambient conditions. For example, it has been observedthat a fresh aqueous solution of concentration, e.g., 1 mg per ml of SQ14,225 at a pH of about 8 becomes substantially less active uponstanding for as little as 30 minutes, and that activity continues todecrease as the solution stands for longer periods. It is believed thatthis loss in activity is mainly the result of dimerization of SQ 14,225occurring at the sulfhydryl end groups, whereby a disulfide is formedwhich is largely inactive as an inhibitor. Since the free sulfhydrylgroup is highly reactive and may be readily oxidized to polar acidicmoieties such as sulfone and sulfoxide groups, it may also be that theobserved in vitro loss of activity of aqueous solutions of SQ 14,225 onstanding is in some part a consequence of one or more such oxidationreactions, with formation of a sulfone or sulfoxide which does notfunction effectively as an inhibitor for ACE.

Such reports of SQ 14,225 clinical testing as are currently available,some of which refer to the compound under the name "Captopril", suggestthat the product is sufficiently stable in the normal gastric andintestinal environments of most patients to be an effective inhibitorfor ACE when administered orally. It is not yet clear, however, whetherthere may be a group of patients for which SQ 14,225 is substantiallyineffective. Because of the high reactivity of the free sulfhydrylgroup, SQ 14,225 could readily form mixed disulfides with serum,cellular proteins, peptides or other free sulfhydryl group-containingsubstances in the gastric or intestinal environments, in addition to thepossibility for dimer formation or oxidative degradation reactions. Amixed disulfide with protein may be antigenic and, indeed, occasionalallergic reactions have been clinically observed. See Gavras, et al.,New England J.Med. 298, 991 (1978). Disulfides and oxidative degradationproducts of SQ 14,225, if formed, may at best be expected to be largelyineffective as inhibitors. It may be postulated accordingly that doseresponse to SQ 14,225 may vary with conditions of administration andamong individual patients. Moreover, in at least some patients, unwantedside effects may occur and maintenance of an effective concentration ofthe inhibitor in the body may be difficult to control.

Thiolester compounds generally are thought to be highly reactive in thatthe thiolester linkage is readly hydrolyzable to a sulfhydryl moiety anda carboxylic moiety. Thiolesters are accordingly often used as activeester intermediates for acylation under mild conditions. Such groups as,e.g., acetylthio have been used as blocking groups in the above citedOndetti, et al. patents. Thiolester intermediates are also postulated tooccur in the biosynthesis of cyclic peptides such as tyrocidin orgramicidin S. See Lipmann, F. in Accounts Chem.Res. 6, 361 (1973).

Thiolester compounds having potent ACE inhibitory activity and oraleffectiveness as anti-hypertensive agents have been disclosed incopending applications Ser. No. 116,950, filed Jan. 30, 1980, which is acontinuation of Ser. No. 941,289, filed Sept. 11, 1978 (now abandoned infavor of its pending continuation Ser. No. 116,950, filed Jan. 30, 1980) and Ser. No. 958,180, filed Nov. 6, 1978 (abandoned in favor of itscontinuation Ser. No. 116,951, filed Jan. 30, 1980 which was abandonedin favor of its continuation, Ser. No. 295,589, filed Aug. 24, 1981which was abandoned in favor of its continuation, Ser. No. 524,204,filed Aug. 18, 1983, which was abandoned in favor of its continuation,Ser. No. 680,541, filed Dec. 11, 1984, which was abandoned in favor ofits pending continuation, Ser. No. 850,055, filed Apr. 10, 1986), bothincorporated herein by reference. The previously disclosed compoundsare: N-[3-(benzoylphenyalanylthio)-2-D-methylpropanoyl]-L-proline (I₅₀≈1-4'N-(2-benzoylphenylalanylthiopropanoyl)-L-proline (I.sub. 50≈4-7×10⁻⁸ M for racemic compound),N-(3-benzoylphenylalanylthiopropanoyl)-L-proline (I₅₀ ≈7×10⁻⁷ M),N-[3-(benzoylphenylalanylthio)-2-D-methylpropanoyl]-L-3,4-dehydroproline,N-(2-benzoylphenylalanylthiopropanoyl)-L-3,4-dehydroproline (I₅₀ ═3×10⁻⁹M for racemic compound), andN-(3-benzoylphenylalanylthiopropanoyl)-L-3,4-dehydroproline. Unlessnoted otherwise, all amino acids are in their L-forms.

Compounds related to SQ 14,225 have been disclosed by Ondetti, et al.,U.S. Pat. Nos. 4,046,889, 4,052,511, 4,053,651, 4,113,715 and 4,154,840.Of interest are disclosed analogs of SQ 14,225 having the five-memberedheterocyclic ring of proline replaced by a four- or a six-membered ring.The inhibitory potencies of such analogs relative to SQ 14,225 are notdisclosed. Substitution of D-proline for L-proline is reported todrastically reduce inhibitory potency of 3-mercaptopropanoyl amino acids(Cushman, D. W., et al., supra).

SUMMARY OF THE INVENTION

Novel inhibitors of ACE are disclosed which have the general formula##STR2## wherein

R is cyclopentylcarbonyl or butoxycarbonyl (also known astert-butyloxycarbonyl);

A is phenylalanyl, glycyl, alanyl, tryptophyl, tyrosyl, isoleucyl,leucyl, histidyl, or valyl whose α-amino group is in amide linkage withR;

R₁ is hydrogen or methyl;

R₂ is proline, 3,4-dehydroproline, D,L-3,4-dehydroproline,3-hydroxyproline, 4-hydroxyproline or L-thiazolidine-4-carboxylic acidwhose imino group is in imide linkage with the ##STR3##

and n is 0 or 1 such that when n=0, R_(l) is methyl. All amino acids arein the L-configuration unless otherwise noted. The disclosed compoundsare inhibitors of ACE and are useful as orally effectiveanti-hypertensive agents.

DETAILED DESCRIPTION OF THE INVENTION

The discovery of ACE inhibitory potency in the compounds of the presentinvention provides a unique approach to the design of inhibitorycompounds. Although many prior art inhibitors are proline derivatives,substitution of other amino acids for proline has also yielded potentinhibitors. Arginine, phenylalanine and alanine are all effectivesubstitutes for proline, so that a trend is not discernible.

The substitution of L-3,4-dehydroproline for proline has been studied inseveral systems. Substitution of L-3,4-ΔPro in the 7 position ofbradykinin yields a bradykinin derivative which has significantlyreduced physiological activity. See Fisher, G. H. et al.,Arch.Biochem.Biophys. 189, 81 (1978). On the other hand, substitution ofL-3,4- ΔPro at the 3, 5 or 9 position in ACE inhibitor BPP_(9a) enhancesits inhibitory activity. In copending application Ser. No. 958,180,applicants found that the compounds having ΔPro, which are disclosed insaid application, have high inhibitory potency and antihypertensiveeffectiveness. However, at present, no rationale can be advanced toexplain the diversity of observed results following substitution of ΔProfor proline. Similarly, no clear picture has emerged of the effects ofother proline derivatives or analogs substituted at various loci on ACEinhibitors.

To date, the effect of the amino acid to the left of the sulfur in theabove-shown formula, has not been determined. It is thought that thisamino acid functions as an additional recognition site for the enzyme.If this is true, it would be expected that a compound with an amino acidhere would be a better inhibitor. It was not known which, if any, aminoacids would be effective in this position and which if any would enhancethe inhibitory activity of a given compound. Applicants have found thatvarious amino acids are effective and that the hydroxyprolines, proline,L-, and D,L-,3,4-dehydroproline, and thiazolidine-4-carboxylic acidderivatives are all effective anti-hypertensive agents and have highinhibitory potency for ACE.

The present invention will be further described by the followingexamples. In these examples, the thin-layer chromatography (TLC) wasperformed using silica gel plates. The numerical solvent systems for usein the TLC procedures are as follows.

(1) is methanol:chloroform, 1:1 (parts by volume). (2) isbenzene:water:acetic acid, 9:1:9 (parts by volume. (3) is aceticacid:water:n-butanol 26:24:150 (parts by volume). (4) isn-butanol:pyridine:acetic acid:water, 15:10:3:12 (parts by volume). (5)is chloroform:methanol:ammonium hydroxide, 60:45:20 (parts by volume).The buffers for paper electrophoresis were: pH 1.9 - formic acid:aceticacid:water, 3:2:25 (parts by volume); pH 5.0 - diethylene glycol:aceticacid:pyridine:water, 100:6:8.5:885 (parts by volume). Thetert-butyloxycarbonyl derivatives of the amino acids are commerciallyavailable.

EXAMPLE 1 Ace Activity Assay

For most experiments described herein, the enzyme was assayed in 0.05 MHepes buffer, pH 8.0 containing 0.1 M NaCl and 0.75 M Na₂ SO₄. Thesubstrate employed was Benzoyl-GlyHisLeu at a final concentration of1×10⁻⁴ M, (K_(m) ≈2×10⁻⁴ M), together with abou of [³H]-Benzoyl-GlyHisLeu (25 Ci/mmole). Enzyme was diluted in the abovebuffer such that 40 μl buffered enzyme was capable of hydrolyzing 13% ofsubstrate in a 15-minute incubation at 37° C. To initiate the assay, 40μl of enzyme and 10 μl of water or inhibitor dissolved in water werepreincubated for five minutes at 37° C. Substrate, 50 μl, was then addedto initiate reaction and the solution was incubated for 15 minutes at37° C. To terminate the reaction, 1 ml of 0.1 M HCl was added, followingwhich 1 ml of ethyl acetate was added. The mixture was agitated on arotary mixer and centrifuged briefly to separate the phases.

An aliquot, 500 μl, of the ethyl acetate layer was transferred to aliquid scintillation vial containing 10 ml of Riafluor, trademark NewEngland Nuclear Corporation, Boston, Mass. For determination of I₅₀values, enzyme activity in the presence of inhibitor at a series ofdifferent concentrations was compared to activity in the absence ofinhibitor. A plot of inhibitor concentration versus percent inhibitionyielded the I₅₀ value.

EXAMPLE 2 Synthesis of 3-acetylthiopropanoyl-L-proline-t-butyl ester

3-acetylthiopropanoic acid, 0.865 g, was dissolved in 2 ml redistilledtetrahydrofuran (THF) and cooled to 0° C. A cooled solution ofdicyclohexylcarbodiimide, 1.2031 g in 2 ml of THF was added, followingwhich a cooled solution of L-proline-t-butyl ester, 1 g, was added. Thereaction mixture was stirred at 0° C. for one hour, then at 4° C.overnight. The reaction mixture was then filtered and the precipitatewas washed with ethyl acetate. Solvents of the filtrates were removedunder reduced pressure in a rotary evaporator. The residue was dissolvedin ethyl acetate which was then washed three times with cold 1 N citricacid, twice with saturated NaCl, twice with cold 1 N NaHCO₃ and threetimes with saturated NaCl. The solution was dried over anhydrous MgSO₄and filtered. The solvent was removed under reduced pressure in a rotaryevaporator at 30° C. yielding a clear colorless oily product inapproximately 87% yield. The product migrated as a single spot in thinlayer chromatography in five solvent systems.

EXAMPLE 3 Synthesis of 3-Mercaptopropanoyl-L-Proline

The product from Example 2, 3-acetylthiopropanoyl-L-proline-t-butylester, 0.5 g, was mixed with 4.5 ml of 5.5 N methanolic ammonia at roomtemperature under nitrogen for one hour to remove the acetyl group. Thesolvent was then removed at 25° C. with a rotary evaporator. After theproduct was taken up in methanol and reevaporated twice more in therotary evaporator, the clear oily residue was dissolved in ethyl ether,washed twice with 5% potassium bisulphate and once with saturated NaCl,dried over MgSO₄ and filtered. Residual solvent was removed in vacuo toyield a clear oily product, migrating as a single spot on thin layerchromatography in three separate solvent systems. The t-butyl esterprotecting group was removed by reaction with trifluoroacetic acid inanisole.

EXAMPLES 4-6

By substituting 2-acetylthiopropanoic acid,3-acetylthio-2-D-methylpropanoic acid, or 3-acetylthio-2-D,L-methylpropanoic acid for the 3-acetylthiopropanoic acid in Example 2and substantially following the procedures of Examples 2 and 3, thefollowing compounds are obtained. By removing the t-butyl esterprotecting group with trifluoroacetic acid in anisole as a first step,the dicyclohexylamine salt can be formed to assist in the resolution ofisomers. The acetyl protecting group can be removed in a second stepusing methanolic ammonia, as described in Example 3.

    ______________________________________                                        Example   Compound                                                            ______________________________________                                        4         2-mercaptopropanoyl-L-proline                                       5         3-mercapto-2-D-methylpropanoyl-L-proline                            6         3-mercapto-2-D,L-methylpropanoyl-L-proline                          ______________________________________                                    

EXAMPLE 7 Synthesis of3-Mercapto-2-Methyl-Propanoyl-L-3,4-Dehydroproline

L-3,4-dehydroproline (Δ³ Pro), 1 mmole, is dissolved in DMF and thesolution is cooled to -15° C. The solution is neutralized by adding 1equivalent of N-ethyl morpholine. In a separate reaction vessel at -10°C., one equivalent of 3-acetylthio-2-methyl-propanoic acid in an equalvolume of DMF is mixed with 1.1 equivalent of 1,1'-carbonyldiimidazole,and the solution is stirred for one hour. The first solution containingΔ³ Pro is mixed with the second, containing 3-acetylthio-2-methylpropanoic acid while maintaining the temperature at -10° C. The combinedsolution is stirred for 1 hour at -10° C. The solution is then allowedto warm slowly to room temperature. The solvent is removed on a rotaryevaporator under reduced pressure at 40° C. Ethyl acetate (25 ml) isadded and the solution is cooled to 0° C. Two ml of 1N citric acid isadded, the two phases are mixed and then allowed to separate. The phasesare separated with a separating funnel, and the organic phase is washedtwice more with 2 ml 1N citric acid, two times with saturated NaCl andfinally dried over anhydrous MgSO₄. The MgSO₄ is removed by filtration,and the solvent is removed with a rotary evaporator. The residue isdissolved and recrystallized from a non-polar solvent such as benzene toyield 3-acetylthio-2-D,L-methylpropanoyl -L-3,4-dehydroproline. When the2-D-methyl isomer is desired, the residue is dissolved in acetonitrile(approximately 3 ml) and the solution is warmed to 40° C. One equivalentof dicyclohexylamine is added, and the solution is allowed to stand atroom temperature overnight. The crystals are colected by filtration andare washed three times with acetonitrile. When further purification isrequired, the material can be recrystallized from isopropanol. Theacetyl protecting group can be removed as in Example 3.

EXAMPLES 8-11

By substituting D,L-3,4-dehydroproline, L-3-hydroxyproline,L-4-hydroxyproline, or L-thiazolidine-4-carboxylic acid for theL-3,4-dehydroproline in Example 7 and substantially following theprocedures of Example 7 the following compounds are obtained.

    ______________________________________                                        Example      Compound                                                         ______________________________________                                         8           3-mercapto-2-D-methylpropanoyl-                                               D,L-3,4-dehydroproline                                            9           3-mercapto-2-D-methylpropanoyl-                                               L-3-hydroxyproline                                               10           3-mercapto-2-D-methylpropanoyl-                                               L-4-hydroxyproline                                               11           3-mercapto-2-D-methylpropanoyl-                                               L-thiazolidine-4-carboxylic acid                                 ______________________________________                                    

EXAMPLE 12

Similarly, by substituting 3-acetylthiopropanoic acid or2-acetylthiopropanoic acid for the 3-acetylthio-2-methyl propanoic acidof Examples 7-11, the L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolioine-4-carboxylicacid derivatives are obtained, following substantially the describedprocedures.

EXAMPLE 13 Synthesis of N.sup.α -[3-(N.sup.α-Tertiary-Butyloxycarbonyl-L-Phenylalanylthio)-2-D-Methylpropanoyl]-L-Proline

A solution of 133 mg of N.sup.α-tertiary-butyloxycarbonyl-L-phenylalanine (N.sup.α -Boc-L-Phe) in 0.5ml redistilled dimethylformamide (DMF) was cooled in an ice-dryice-acetone bath at -20° C. To this solution was added a cold solutionof 87 mg of 1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution wasstirred at -10° C. for two hours and then was added to a cold solutionof 119.5 mg of 3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMFwhich was neutralized with N-ethyl morpholine. The reaction mixture wasstirred at -10° C. for an additional hour and then slowly warmed to roomtemperature. The solvent was removed under reduced pressure at 40° C.and ethyl acetate was added to the residue. The mixture was cooled in anice bath and washed with 0.1 N HCl and then three times with saturatedNaCl solution. The solvent was removed with a rotary evaporator afterdrying over anhydrous MgSO₄. The product was purified by liquidchromatography on Sephadex G-10 using a 1.2 cm by 95 cm column andeluted with THF:isopropanol, 3:7 (parts by volume). The peak fractionswere pooled and the solvent removed under reduced pressure yielding 165mg of the named product. This product was found to be homogeneous usingpaper electrophoresis at pH 5.0 and using TLC with solvent systems 1, 2and 3.

EXAMPLES 14-21

By substituting N.sup.α -Boc-glycine, N.sup.α -Boc-alanine, N.sup.α-Boc-tryptophan, N.sup.α -Boc-tyrosine, N.sup.α -Boc-isoleucine, N.sup.α-Boc-leucine, N.sup.α -Boc-histidine or N.sup.α -Boc valine for theN.sup.α -Boc-L-Phe in Example 13 and substantially following theprocedures of Example 13, the following compounds are obtained.

    ______________________________________                                        Example                                                                              Compound                                                               ______________________________________                                        14     N.sup.α --[3-(N.sup.α --butyloxycarbonylglycylthio)-2-D           -                                                                             methylpropanoyl]-L-proline                                             15     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-tryptophylth           io)-2-D-                                                                      methylpropanoyl]-L-proline                                             16     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-tyrosylthio)           -2-D-                                                                         methylpropanoyl]-L-proline                                             17     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-isoleucylthi           o)-2-D-                                                                       methylpropanoyl]-L-proline                                             18     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-leucylthio)-           2-D-                                                                          methylpropanoyl]-L-proline                                             19     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-histidylthio           )-2-D-                                                                        methylpropanoyl]-L-proline                                             20     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-valylthio)-2           -D-                                                                           methylpropanoyl]-L-proline                                             21     N.sup.α --[3-(N.sup.α --butyloxycarbonyl-L-alanylthio)-           2-                                                                            D-methyl propanoyl)-L-proline)                                         ______________________________________                                    

EXAMPLE 22

Similarly, the L-3,4-dehydroproline, D,L,3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline, and L-thiazolidine derivativesare obtained by substituting the products of Examples 3-12 for the3-mercapto-2-D-methyl-propanoyl-L-proline in Examples 13-21 andsubstantially following the procedure of Example 13.

EXAMPLE 23 Synthesis of N.sup.α -cyclopentylcarbonyl-L-phenylalanine.

A cool solution of 2.06 gm of dicyclohexylcarbodiimide in 10 ml ofdichloromethane was added to a solution of 1.4114 gm ofcyclopentanecarboxylic acid in 5 ml of dichloromethane at -5° C. It wasfollowed by the addition of 4.28 gm of L-phenylalanine benzoyl estertouluenesulfonate salt in 10 ml of DMF which was neutralized with 1.36ml of N-ethyl morpholine. The reaction mixture was stirred at 0° C. forone hour and then at room temperature for three hours. Dicyclohexylureawas removed by filtration and 50 ml of ethyl acetate was added to thefiltrate. The organic phase was washed until neutral, dried overanhydrous MgSO₄ and filtered. The solvent was removed with a rotaryevaporator. The residue was crystallized from isopropanol and hexaneyielding 2.35 gm of white crystals having a melting point of 88-89° C.Elemental analysis of these crystals yielded the following.

Calculated: C=75.19; H=7.17: N=3.9855.

Found: C=74.96; H=7.17; N=4.09.

The benzyl ester was removed by hydrogenolysis with 2 gm of 10%palladium on carbon in absolute alcohol. The catalyst was removed byfiltration and the ethanol was removed by a rotary evaporator. Theresidue was crystalized from ether and hexane yielding 1.15 gm whitecrystals of the named product having a melting point of 107°-108° C.Elemental analysis of these crystals yielded the following.

Calculated: C=68.94; H=7.33; N=5.36.

Found: C=68.90; H=7.32; N=5.34.

The product was found to be homogeneous using paper electrophoresis atpH 1.9 and at pH 5.0 and using TLC with solvent systems, 1, 2 and 3. Thenamed product may be abbreviated as N.sup.α -cpc-L-Phe.

EXAMPLE 24 Synthesis of N.sup.α -[3-(N.sup.α-cyclopentylcarbonyl-L-phenylalanylthio)-2-D-methylpropanoyl]-L-proline

A solution of 52.5 mg of the compound from Example 22 in 0.5 mlredistilled dimethylformamide (DMF) was cooled in an ice-dry ice-acetonebath at -20° C. To this solution was added a cold solution of 34 mg of1,1'-carbonyldiimidazole in 1.0 ml of DMF. The solution was stirred at-10° C. for two hours and then mixed with a cold solution of 45.6 mg of3-mercapto-2-D-methylpropanoyl-L-proline in 1 ml of DMF which wasneutral N-ethyl morpholine. The reaction mixture was stirred at -10° C.for an additional hour and then slowly warmed to room temperature. Thesolvent was removed under reduced pressure at 40° C. and ethyl acetatewas added to the residue. The mixture was cooled and washed with 0.1 NHCl and then three times with saturated NaCl solution. The solvent wasremoved with a rotary evaporator after drying over anhydrous MgSO₄. Theproduct was purified by LH-20¹ column chromatography using a 1.2 cm by95 cm column and eluted with isopropanol. The peak fractions were pooledand the solvent was removed under reduced pressure yielding 60.5 mg ofthe named product. This product was found to be homogeneous using TLC insolvent systems 1, 2, 3 and 5.

EXAMPLES 25-32

By substituting the benzoyl ester toluenesulfonate salts of glycine,L-alanine, L-tryptophan, L-tyrosine, L-isoleucine, L-leucine,L-histidine or L-valine for the L-Phe salt in Example and substantiallyfollowing the procedures of Examples 22 and 23, the following compoundsare obtained.

    ______________________________________                                        Exam-                                                                         ple   Compound                                                                ______________________________________                                        25    N.sup.α --[3-(N.sup.α --cyclopentylcarbonylglycylthio)-2          -D-                                                                           methylpropanoyl]-L-proline                                              26    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-tryptophyl          thio)-2-D-                                                                    methylpropanoyl]-L-proline                                              27    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-tyrosylthi          o)-2-D-                                                                       methylpropanoyl]-L-proline                                              28    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-isoleucylt          hio)-2-D-                                                                     methylpropanoyl]-L-proline                                              29    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-leucylthio          )-2-D-                                                                        methylpropanoyl]-L-proline                                              30    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-histidylth          io)-2-D-                                                                      methylpropanoyl]-L-proline                                              31    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-valylthio)          -2-D-                                                                         methylpropanoyl]-L-proline                                              32    N.sup.α --[3-(N.sup.α --cyclopentylcarbonyl-L-alanylthio          )-2-D-                                                                        methylpropanoyl]-L-proline                                              ______________________________________                                    

EXAMPLE 33

Similarly, the L-3,4-dehydroproline, D,L-3,4-dehydroproline,L-3-hydroxyproline, L-4-hydroxyproline and L-thiazolidine-4-carboxylicacid derivatives are obtained by substituting the products of Examples3-12 for the 3-mercapto-2-D-methylpropanoyl-L-proline in Examples 24-32and substantially following the procedure of Example 24.

EXAMPLE 34

The inhibitory potency of N.sup.α -[3-(N.sup.α-Boc-L-Phe-thio)-2-D-methylpropanoyl]-L-proline (A) and N.sup.α-cpc-L-Phe-thio)-2-D-methylpropanoyl]-L-proline (B) in vitro wasmeasured in the assay system described in Example 1. The enzymepreparation was ACE purified from human urine as described by Ryan, J.W., et al., Tissue and Cell 10, 555 (1978). The I₅₀ for (A) was found tobe 3.4×10⁻⁸ M and for (B), 7.5×10⁻⁹ M.

EXAMPLE 35 Oral effectiveness of N.sup.α -[3-(N.sup.α-cyclopentylcarbonyl-L-phenylalanylthio)-2-D-methylpropanoyl]-L-proline

Rats (190-290 g body weight) were fasted overnight and then anesthetizedwith intraperitoneal pentobarbital, 50-60 mg/kg. Tracheostomy wasperformed and the animals were ventilated mechanically. A cannula wasinserted into a femoral vein for injection of angiotensin I, and asecond cannula was inserted into a common carotid artery for directmeasurement of arterial blood pressure. Heparin, 1,000 units, wasinjected via the femoral vein to prevent coagulation. Blood pressure wasmeasured with a pressure transducer connected to a polygraph. The ratswere injected with 400 ng/kg of angiotensin I in 20 μl of 0.9 g % NaCl;an amount of angiotensin I sufficient to raise mean arterial bloodpressure by 35 mm Hg. After the responsiveness of a given rat toangiotensin I was established, the named compound at 23 μmole/kg (drugdissolved in 0.15 ml of H₂ O plus 10 μl of 1 N NaHCO₃ ), was given via astomach tube. At timed intervals, the effects of 400 ng/kg ofangiotensin I on mean arterial blood pressure were tested. Results areshown below:

    ______________________________________                                        Time After Oral Blood Pressure Response to                                    Administration  400 ng/kg of Angiotensin I                                    (Minutes)       (% of Control)                                                ______________________________________                                        -5              100      (35 mm Hg)                                           +2              71                                                            6               63                                                            16              49                                                            23              43                                                            37              43                                                            48              40                                                            64              26                                                            71              26                                                            84              37                                                            96              37                                                            109             40                                                            124             54                                                            140             51                                                            157             63                                                            171             77                                                            ______________________________________                                    

EXAMPLE 36 Intravenous effectiveness of N.sup.α -[3-(N.sup.α-tert-butyl-oxycarbonyl-L-phenylalanylthio)-2-D-methylpropanoyl]-L-proline

Anesthetized rats were prepared as described in Example 35. After theresponsiveness of a given rat to angiotensin I was established, thenamed compound, at 2 μmol/kg, in a volume of 15 μl of 0.01 N sodiumbicarbonate, was injected via a femoral vein. At timed intervals, theeffects of angiotensin I, 400 ng/kg, on mean arterial blood pressurewere tested. Results are shown below:

    ______________________________________                                        Time after intravenous                                                                          Blood pressure response to                                  administration    400 ng/kg Angiotensin I                                     (minutes)         (% of control)                                              ______________________________________                                        -5                100     (32 mm Hg)                                          +0.5              0                                                           3                 19                                                          9                 19                                                          13                22                                                          18                31                                                          22                31                                                          34                50                                                          47                63                                                          65                78                                                          103               75                                                          113               94                                                          ______________________________________                                    

EXAMPLE 37 Oral effectiveness of N.sup.α -[3-(N-.sup.αtert-butyloxycarbonyl-L-phenylalanythio)-2-D-methylpropanoyl]-L-proline

The procedure of Example 35 was followed. The Angiotensin I response was37 mm Hg and the oral dose administered was 20 μmol/kg. Results areshown below:

    ______________________________________                                        Time after intravenous                                                                          Blood pressure response to                                  administration    400 ng/kg angiotensin I                                     (minutes)         (% of control)                                              ______________________________________                                        -5                100     (37 mm Hg)                                          +3                100                                                         11                43                                                          22                27                                                          29                24                                                          34                22                                                          51                19                                                          ______________________________________                                    

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

What is claimed is:
 1. A new compound having the formula ##STR4##wherein R is cyclopentylcarbonyl or tert-butyloxycarbonyl;A isL-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl,L-leucyl, L-histidyl, or L-valyl whoseα-amino group is an amide linkagewith R; R₁ is hydrogen or methyl; R₂ is L-proline, L-3,4-dehydroproline,D,L-3,4-dehydroproline, L-3-hydroxyproline, L-4-hydroxyproline orL-thiazolidine-4-carboxylic acid whose imino group is in imide linkagewith the ##STR5## and, n is 0 or 1, such that when n=0, R₁ is methyl. 2.A compound of claim 1 wherein R₂ is L-proline.
 3. A compound of claim 1wherein R₂ is L-3,4-dehydroproline.
 4. A compound of claim 1, 2 or 3wherein R₁ is cyclopentylcarbonyl, A is L-phenylalanyl, n is 1 and R₁ ismethyl.
 5. A compound of claim or 1, 2 or 3 wherein R istertbutyloxycarbonyl, A is L-phenylalanyl, n is 1 and R1 is methyl.
 6. Acompound of claim 1, 2 or 3 wherein A is L-phenylalanyl, L-tryptophyl,L-tyrosyl or L-histidyl.
 7. A compound of claim 1, 2 or 3 wherein A isglycyl, L-alanyl, L-isoleucyl, L-leucyl or L-valyl.
 8. A method forinhibiting angiotensin converting enzyme in vivo comprisingadministering an effective dose of a compound having the formula##STR6## wherein R is cyclopentylcarbonyl or tert-butyloxycarbonyl;A isL-phenylalanyl, glycyl, L-alanyl, 1-tryptophyl, L-tyrosyl, L-isoleucyl,L-leucyl, L-histidyl, or L-valyl whoseα-amino group is in amide linkagewith R; R₁ is hydrogen or methyl; R₂ is a residue of L-proline,L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline,L-4-hydroxyproline or L-thiazolidine-4-carboxylic acid whose imino groupis in imide linkage with the ##STR7## and, n is 0 or 1, such that whenn=0, R₁ is methyl.
 9. The method of claim 8 wherein R₂ is L-proline. 10.The method of claim 8 wherein R₂ is L-3,4-dehydroproline.
 11. The methodof claim 8, 9 or 10 wherein R is cyclopentycarbonyl, A isL-phenylalanyl, n is 1 and R₁ is methyl.
 12. The method of claim 8, 9 or10 wherein R is tertbutyloxycarbonyl, A is L-phenylalanyl, n is 1 andR_(l) is methyl.
 13. The method of claim 8, 9 or 10 wherein A isL-phenylalanyl, L-tryptophyl, L-tyrosyl or L-histidyl.
 14. The method ofclaim 8, 9 or 10 wherein A is glycyl, L-alanyl, L-isoleucyl, L-leucyl orL-valyl.
 15. A method for reducing blood pressure in vivo comprisingadministering an effective dose of a compound having the formula##STR8## wherein R is cyclopentylcarbonyl or tertbutyloxycarbonyl;A isL-phenylalanyl, glycyl, L-alanyl, L-tryptophyl, L-tyrosyl, L-isoleucyl,L-leucyl, L-histidyl, or L-valyl whose α-amino group is in amide linkagewith R; R₁ is hydrogen or methyl; R₂ is a residue of L-proline,L-3,4-dehydroproline, D,L-3,4-dehydroproline, L-3-hydroxyproline,L-4-hydroxyproline or L-thiazolidine -4-carboxylic acid whose iminogroup is in imide linkage with the ##STR9## and, n is 0 or 1, such thatwhen n=0, R is methyl.
 16. The method of claim 15 wherein R₂ isL-proline.
 17. The method of claim 15 wherein R₂ isL-3,4-dehydroproline.
 18. The method of claim 15, 16, or 17 wherein R iscyclopentylcarbonyl, A is L-phenylalanyl, n is 1 and R₁ is methyl. 19.The method of claim 15, 16 or 17 wherein R₁ is tertbutyloxycarbonyl, Ais L-phenylalanyl, n is 1 and R₁ is methyl.
 20. The method of claim 15,16 or 17 wherein A is L-phenylalanyl, L-tryptophyl, L-tyrosyl orL-histidyl.
 21. The method of claim 15, 16 or 17 wherein A is glycyl,L-alanyl L-isoleucyl, L-leucyl or L-valyl.